MXPA98008006A - Novedoso transcription factor, tfiib, of candida albicans, sequence of nucleic acids that codify thereof, and methods of selective classification of growth inhibitors of candida albic - Google Patents

Abstract

The present invention relates to a novel transcription factor of Candida albicans, the TFIIB, to a nucleic acid sequence encoding the TFIIB, and to methods for the selective classification of growth inhibitors of Candida albicans by means of the TFIIB as an objective target.

Description

NOVEDOSO TRANSCRIPTION FACTOR - TFIIS, DEL Candida aZLbi.ca.ns, SEQUENCE OF NUCLEIC ACIDS THAT CODIFY THE SAME, AND SELECTIVE CLASSIFICATION METHODS OF INHIBITORS OF THE GROWTH OF Candida a bieaps FIELD OF THE INVENTION The invention relates, in general, to transcription factors and methods for the selection of antifungal agents. The invention was made, in part, using government funds, NIH grant No. GM46498, and therefore the government of the The United States has certain rights over the invention.

BACKGROUND OF THE INVENTION The yeast Candi da albi can s (C. albi can s) is one of the most penetrating fungal pathogens in humans. It has the ability to opportunistically infect a diverse spectrum of compromised hosts, and to invade very diverse tissues in the human body. In many cases you can avoid treatment with REF. : 28479 antibiotics and the immune system. Although the Candi da albi can s is a member of the normal flora of the mucous membranes of the tracts r esp ira-chorio, gastrointestinal, and female genital, in those sites may have dominance and be associated with pathological conditions. Sometimes it produces a progressive systemic disease in weakened patients - or immunosuppressed patients, particularly if the cell-mediated immunity is impaired. Sepsis may occur in patients with compromised cellular immunity, for example those who receive cancer chemotherapy or those with lymphoma, AIDS, or other conditions. The Candi da can produce invasion into the bloodstream, thrombophlebitis, endocarditis, or infection of the eyes and virtually any organ or tissue when it is introduced intravenously, for example, via tubes, needles, narcotic abuse, etc. It has been shown that the Candi gives to lebi cans is diploid with balanced lethal and therefore probably does not pass through a sexual phase or meiotic cycle. It seems that this yeast is capable of changing, "at a high Frequency and in an exponential and reversible way, between at least seven general phenotypes. It has been shown that changes occur not only in standard laboratory strains, but also in strains isolated from the mouth of healthy individuals. Nystatin, ketoconazole, and amphotericin B are drugs that have been used to treat oral and systemic infections by Candi da. However, nystatin administered orally is limited to treatment within the intestines and is not applicable to systemic treatment. Some systemic infections are susceptible to treatment with ketoconazole or amphotericin B, but these drugs may not be effective in that treatment unless they are combined with additional drugs. Amphotericin B has a relatively narrow therapeutic index and there are numerous undesirable side effects as well as toxicity, even at therapeutic concentrations. Although ketoconazole and other de-azole antifungal agents exhibit significantly low toxicity, its mechanism of action, the inactivation of the prosthetic group of cytochrome P450 in certain enzymes, some of which are found in "humans, preclude use in patients who are simultaneously receiving other drugs that are metabolized by the body's cytochrome P450 enzymes. These compounds are emerging and may pose a serious problem in the future. effective treatment of opportunistic infections caused by canitis to bi cans. Therefore, an object of the invention is to provide selection tests. for the identification of potential inhibitors of growth of Candi gives to lbi can s. Another objective of the invention is to provide screening assays and identify potential inhibitors of the growth of Candi da albi cans, which are based on the inhibition of transcription in -20 this body. The synthesis of mRNA in eukaryotes requires d-e RNA polymerase II and additional transcription factors, some of which are general and act mostly, if not all, as promoters, and others of which they confer specificity and control. Five general factors, a, b, d, e, and g, have been purified to have homogeneity from yeast S. c ere vi si a e, and have been identified as counterparts to rat or human factors, TFIIE, TFIIH, TFIID, ~ TFIIB and TFIIF, respectively. These factors bind to a promoter-r "in a complex with RNA polymerase II to initiate transcription.Binding studies have shown that the order of initiation complex assembly on promoter DNA starts with factor d (TFIID ), is followed by the f-actor e (TFIIB), and then by the polymerase and the remaining factors, however, factors b (TFIIH), e (TFIIB), e (TFIIB) and g (TFIIF), are they link directly to polymerase II, and as many as four of the "five factors can be linked to the polymerase in a holoenzi before promoter binding. The functional significance of the interactions revealed by the binding studies is not clear - because only a small percentage of the initiation complexes can give rise to transcripts. Many aspects of the certification by RNA polymerase II are conserved between yeast and higher eukaryotes. For example, there is extensive similarity in the amino acid sequences between the larger subunits of the yeast, de-Drosophila and mammalian polymerase. Other components of the transcription apparatus, such as TATA binding and enhancer binding factors, are in some cases interchangeable between binding or transcription systems, in vitro, yeast and mammals, however, there are significant differences between them. Two systems The TATA elements are located from -40 to 120 or more base pairs upstream of the initiation site of a S. cerevisiae promoter, and where these elements are presented, they are required for the Candi gn ti expression. The fact that C. albicans genes function in S. cervisvisiase suggests that it also uses the spacing of 40 to 120 base pairs between the TATA element and the initiation site, in contrast, mammalian TATA elements. (as well as those of S. pombe) and the transcription start sites, they are separated by only 25 to 30 base pairs, and the elimination of a TATA element does not always reduce the frequency of the initiation of transcription, although it may alter the initiation site. There are also varying degrees of homology between the transcription factor sequences of yeast and mammalian sources. Human and mammalian TFIIBs have an amino acid sequence identity of 50 to 60%, and they are not interchangeable species to support cell growth. Some of the multiple subunit factors, such as RNA polymerase II, TFIIF and TrTTD, contain different numbers of subunits in humans and in yeast. The molecular weights of the corresponding polypeptides differ in humans and in yeast, and sequences are present in a given factor of the yeast that are not present in their human counterpart, and vice versa. The operative substitution of the same transcription factor, in transcription systems of different strains of yeast, can not be predicted. This is true even though There is a high degree of identity of the amino acid sequences between some transcription factors of different yeast strains. For example, the ability of a given transcription factor to support transcription, efficient and accurate, in a heterologous yeast strain can not be predicted. Li et al. (1994), Science 263: 805) analyzed the interchangeability of the transcription factors of S. c e and vi if a and e of 5. po-n.be, in vitro, and report that many components of S. cerevi sl e can not individually substitute transcription factors a, e, or RNA polymerase II, of S_. pomb? j_ but what combinations of these components were effective. In one case, active transcription could not be reconstituted when the TFIIB derived from 5. c ere vi sl a e was the only substitution in a set of factors with reduction or depletion of TFIIB, from 5. pombe. A combination of TFIIB-RNA polymerase II, from S. cer-evi if a, was able to substitute, which indicated that the functional interaction of these two components is not only important, but also the activity can be dependent on the specific determinants of the species that can not be complemented by any other component derived from a different organism- The imprédecibilidad in the realization of the substitutions of a given factor between different strains of yeast is also evident in that these substitutions are not reciprocal; that is to say, the substitutions of fractions of the 5. pombe in a system for the transcription of S. cererisiae are less effective than inverse substitutions (Li et al., supra). The yeast Candida to bi can s differs from most yeast strains in that it does not use the same genetic code as the one used by most organisms, be it mammal or yeast. Santos et al. (1995, Nucleic Acids Research, 23: 1481) reports that the CUG codon, which in the universal code is read as a leucine, is decoded as a serine in the Candi da. Therefore, any CUG codon that is decoded in the Candi gives the bi can s as a serine would be decoded as a leucine in the S. I saw if it was transformed. Any gene that contains a CUG codon would therefore be translated as different amino acid sequences in the Candida albicans and in the 5. cerevisiae. That mistranslation can produce an inactive protein, since the amino acids serine and leucine have markedly different chemical properties and it is known that serine is an essential residue in the active site of some enzymes. The replacement of leucine by serine in CUG-encoded residues is a serious problem in the use of many reporter systems (for example, Beta-galactosidase, Cloranf-en-acetyl trans-fe-asa, Flux) in Candida albi cans. Previous experiments have shown that the translation of CUG by Candida, as a serine instead of leucine, has often resulted in the production of inactive reporter proteins. Another objective of the invention is to provide a selection test for the selective inhibition of growth and / or viability of Candida albicans. Still another object of the invention is to provide a molecular target for the inhibition of transcription or initiation of transcription of Candida albicans.

BRIEF DESCRIPTION OF THE INVENTION The invention comprises a recombinant nucleic acid containing a nucleic acid sequence encoding the TFIIB of Candida albicans. The invention also comprises a vector containing a nucleic acid sequence encoding the TFIIB of Candi da albi cans, and a transformed host cell containing a nucleic acid sequence encoding "for the TFIIB of Candida albicans. comprises a recombinant polypeptide comprising the TFIIB of Candida albicans, and a fragment of the TFIIB of Candida albicans, and the fragment is characterized in that it inhibits the biological activity of the TFIIB of Candida albicans at the initiation of transcription, or prevents the growth of Candida albícans . The invention also comprises a method for producing TFIIB from recombinant Candida albicans, which comprises culturing a host cell transformed with a nucleic acid encoding the TFIIB of Candida albicans under-conditions sufficient to allow the expression of the nucleic acid encoding the TFIIB of Candida albicans, and isolate the TFIIB from Candida albicans. The invention also comprises a method of selective classification for identifying a growth inhibitor of Candida albicans, which comprises detecting the inhibition of transcription of mRNA in an in vitro transcription assay comprising a DNA standard, RNA polymerase II, TFIIB of Candida albicans, recombinant, and a candidate inhibitor, wherein the production of a transcribed mRNA, complementary to the DNA standard, occurs in the absence of the candidate inhibitor. Preferably the assay will also include TBP of -Candida albicans. The invention also comprises a method of selective classification to identify a growth inhibitor of Candida albicans, which comprises detecting, in the presence of a candidate inhibitor, the inhibition of the formation of a complex comprising TFIIB and TBP of Candida albicans, recombinant , of the DNA pattern, where in the In the absence of the candidate inhibitor, complex formation occurs. The invention also comprises a method of selective classification for the identification of a growth inhibitor of Candida albicans, which comprises detecting, in the presence of a candidate inhibitor, the inhibition of the formation of a complex comprising TFIIB of Candida albicans and TBP of Candida albicans, where, in the absence of the candidate inhibitor, complex formation occurs. Preferably, the complex will include a DNA standard. The invention also comprises a method of selective classification for the identification of the growth of Candida albi cans, which comprises detecting, in the presence of a candidate inhibitor, the inhibition of the formation of a complex comprising RNA polymerase II, TBP of Candi da albi ans, and TFIIB of Candida albicans, where, in the absence of the candidate inhibitor, complex formation occurs. Preferably, the complex may also include a DNA standard; and RNA polymerase of Candida albicans.

In the above-mentioned screening methods, detection can be performed in the presence of a plurality of candidate inhibitors. In the methods of examination of the invention, which involve the examination of a plurality of candidate inhibitors, the plurality of inhibitors can be examined together in a single assay or individually by the use of single, simultaneous, multiple detection steps. The invention also comprises a method for preventing the growth of the Candi da lbi cans in culture, which comprises contacting the culture with an inhibitor that selectively inhibits the biological activity of the TFIIB of the Candi da albí cans. The invention also comprises a method for preventing the growth of Candi da lbi cans in a mammal, which comprises contacting the mammal with an inhibitor that selectively inhibits the biological activity of the TFIIB of Can di da albí cans. As used herein, "inhibition" refers to a reduction in the measured parameter, either growth or viability of Candida albicans, the transcription mediated by the TFIIB of Candida albicans, or the formation of a transcription complex of the TFIIB of Candida albícans. The amount of that reduction is measured in relation to a pattern (control). Due to the multiple interactions of the TFIIB of Candida albicans at the initiation of transcription, the target or target product for detection varies with respect to the particular test test employed. Three preferred detection products, presented in this description, are; a) recently transcribed mRNA, b) a DNA-TFIIB complex, and c) a TBP-TFI IB-RNA polymerase complex. "Reduction" is de-fine herein as a decrease of at least 25% relative to a control, preferably at least 50%, and most preferably at least 75%. As used herein, "growth" refers to the normal growth pattern of Candida albicans, i.e., a cell doubling time of 60 to 90 minutes. "Feasibility" refers to the ability of Candida albícans to survive in the crop for 48 hours.

"Biological activity" refers to the ability of TFIIB to form a transcription complex with a DNA pattern or other proteins of the transcription complex, or to interact with other components of transcription, in order to allow the initiation of transcription. "DNA standard" refers to double-stranded DNA and, where indicated, by-the particular binding assay, single-stranded DNA, at least a length of 10 nucleotides, which may be super-fungally negatively, possess a promoter region, and contains a TATA consensus region of the yeast, upstream of the promoter. DNA templates useful herein preferably contain a TATA sequence that is localized from 40 to 120 or more base pairs upstream of the initiation site (distance measured from the first T of the TATA element to the more initiated 5 'site). ). A particularly efficient DNA standard for use in the methods of the invention involving transcription is devoid of guanosine residues, and therefore a cartridge is preferred.

"G-less" or "G-lower" (Sa dago and Roeder, 1985, PNAS 82: 4394-4398). "mRNA transcript" refers to a full-length transcript, as well as to truncated transcripts, oligonucleotide transcripts and dinucleotide RNAs. "Complex formation" refers to the binding of TFIIB to other transcription factors (i.e., protein-protein binding) as well as to the binding of TFIIB to a DNA standard; this link will, of course, be a non-covalent association. Other features and "advantages of the invention will become apparent from the description, from the preferred embodiments thereof, from the drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 presents the nucleotide and amino acid sequences of the TBP transcription factor of Candi da albi cans.

Figure 2 presents sequences of nucleotides and amino acids of the transcription factor TFIIB of Can di da albí can s.

DESCRIPTION OF THE INVENTION The invention is based on "the discovery of a new protein, the TFIIB of Candi da albi can s, and the isolation of the -DNA recombinant coding for the transcription factor TFIIB of Can di da albi can s. TFIIB is essential for the viability of the cell, it is expected that a compound that blocks the biological activity of the protein has fungicidal properties.Therefore, the invention is also based on the development of assays for the examination of TFIIB inhibitors.

Isolation and Characterization of TBP and TFIIB genes from Candi da albi cans Given the unpredictability with respect to the operational substitutions of a given transcription factor between different strains of yeast, it can not be assumed which strategies will work for the cloning of the gene that encodes a given transcription factor, which are based on the function of the factor, such as genetic complementation. Other cloning strategies, which do not require functional complementation, such as those based on homology at the nucleic acid level, can be used in an attempt to circumvent a requirement for the factor function. For example, Southern hybridization of specific sequences for a library carried on E. col i and PCR amplification of potential and highly homologous regions of a gene are two strategies that have been successfully used to clone homologous genes from different organisms. To investigate the likelihood of success of these methods or similar methods, a Southern hybridization experiment was carried out where a radiolabeled probe containing the entire TBP coding sequence (SPT15) of 5. cerevisi ae was hybridized in genomic DNA preparations of S. c evi if a and Can di gives albí can s that were digested with several restriction enzymes. At low accuracy (0.2 X SSC, hybridization at room temperature) the probe efficiently hybridized, as expected, to bands of restriction in the digested material of S. cerevisiae, but did not detectably hybridize to any restriction band of the digested material of Candida albicans. The sequences of the Saccharomyces could be detected in a autoradiograph with less than 10 minutes of exposure. By contrast, even with one week of exposure, there was no detectable hybridization of the same probe to the DNA of Candida albi cans. The conclusion of these observations is that it is probable that the DNA sequence of the Candida albicans gene has diverged quite significantly from the S sequence. cere vi siae, and therefore it is unlikely that Southern hybridization, PCR strategies or other cloning methods based on the hybridization of complementary nucleic acid sequences will lead to the cloning of the SPT15 homolog of Candi da albi cans. The approach used to clone the TFIIB homolog of Candida albicans involved the genetic complementation of the mutant strains of 5. cerei-isiae. A library of genomic sequences of Candida albicans was introduced in a strain of? . I saw if it contained a mutated TFIIB gene (SUA7). This mutant strain was able to grow at 30 ° C, but was not viable at 37 ° C, due to the temperature-sensitive mutation, in the TFIIB gene. Following the transformation of the library into the strain, the cells were cultured at 37 ° C, and the colonies that grew at this non-allowable temperature were further studied as potential carriers of a Candi da homologue to the bi-tiredness of the defective gene. The plasmids were purified from several isolates that were viable at 37 ° C. After the candidate clones were isolated by growth at the non-pe-en-i temperature, the plasmid was recovered from the library, from the cell, and the analysis was re-done to confirm that the C. bi can s in the plasmid were replacing the gene of S. c ere vi si ae. The subclones of the sequences from C. to Ibs are constructed through standard cloning methods, and the minimum DNA sequence of Can di da was ordered using standard methods. Digestion of these plasmids revealed a common DNA fragment of approximately 1.35 kb in length, which when pRS316 was subcloned tried to complement multiple independent SUA7 strains of S. cerevisiae. This DNA fragment was ordered in sequence, and confirmed the homology of the SUA7 sequence of C. albicans with the SUA7 of S. cerevisiae. The sequence of nucleotides encoding the TBP of Candida albicans and the predicted amino acid sequence of the encoded protein are as shown in Figure 1 (SEQ ID NOs: 1 and 2). The sequence ---- of nucleotides encoding the TFIIB of Candida alblasts and the predicted amino acid sequence of the encoded protein are presented in Figure 2 (SEQ ID NOS: 3 and 4).

Methods for the Examination of Potential Growth Inhibitors and / or Viability of Candida albicans Because TFIIB is essential for the initiation of transcription of the TFIIB gene of Candida albicans, recombinant, and the recombinant protein encoded by this gene, they can be used in test assays for inhibitors of growth and viability of Candida albicans. The tests of selective classification of inhibitors of the growth and viability of the Candi gives to lbi cans. The selective classification assays of this invention detect the inhibition of the initiation of transcription mediated by the TFIIB of the Candi al to the b c an s, either through the measurement of the inhibition of transcription, the initiation of transcription, or the formation of the initiation complex, or by assaying the formation of a protein / DNA or protein / protein complex. 10 EXAMPLE 1 Selective Classification of Inhibitors d-e Transcription a) Components of the Test of the Transcription. i * An in vitro transcription assay consisting of the minimum components can be used necessary to synthesize a mRNA transcript from a DNA standard, to classify or analyze the inhibition of mRNA production. The elements of that assay consist of, a) a DNA standard, b) RNA polymerase II, c) TFIIB of Can di da albi can s, recombinant, and d) a TBP that is preferably the «TBP del Candi gives the bi can s. To increase the efficiency of transcription, additional components of the transcription complex may be included, as desired; for example, TFIIE, 5 TFIIF, TFIIH, etc. Parvin and Sharp (Cell 73, 533-540, 1993) have reconstituted genetic transcription in vi tro with a minimal reaction containing a DNA standard, RNA polymerase II, TFIIB, and TBP. For the * 10 efficient transcription under minimal conditions, the DNA standard (a) is supercoiled, and (b) possesses a promoter region containing a consensus region of TATA. Additionally, Lúe et al. (Science 246, 661-664, 1989) have determined that transcription can be determined in the most efficient manner with a DNA pattern devoid of guanosine residues (a G-minus or G-lower cartridge). The promoter dependency is demonstrated by the signal loss, when the promoter sequences lacking plasmids are used as a pattern. The correct initiation is demonstrated by the production of a band with a mobility that is consistent with the expected product size on polyacrylamide based electrophoresis gels denaturing.

As mentioned above, the TFIIB of Candida albicans forms a complex of initiation of transcription with RNA polymerase II. Therefore, it is desired that a transcription assay i_n vi tro, according to the invention, contain RNA polymerase II. Although it is possible to perform a selective inhibitor assay, using RNA polymerase II from a yeast strain, different from Candida albícans, for example S. cerevisiae, it is most desirable to use a homologous assay in which the components of the complex of transcript of Candida albicans. A method for the purification of RNA pol III from 5. cerevisiae is described in Edwards et al. (Proc. Nati, Acad. Sci. USA 87: 2122-2126 (1990)). Alternatively, RNA polymerase II Candi da albi cans, highly purified, was provided as follows. RNA polymerase activity was measured in reactions containing 50 mM Tris-Cl, pH 7.9 (4 ° C), 50 mM (NH,) 2 S0, 2.5 M MnCl2, 0.1 mM EDTA, 5 M ' DTT, 100 μg / ml of BSA, 0.6 mM of ATP, CTP and GTP, 25 μM of UTP (2.5 μCi) [a-32P] UTP and 100 μg / ml of heat denatured calf thymus DNA, in one volume end of 50 μl. The reactions were incubated for 60 minutes at 30 ° C and finalized by the addition of 50 μl of 15% trichloroacetic acid (w / v). The acid insoluble radioactivity was collected by filtration through glass fiber filters and quantified by liquid scintillation spectrophotometry. One unit of activity of the RNA polymerase catalyses the incorporation of 1 pmol of UTP in the acid-soluble material, in 60 minutes, under the conditions described above. Can di da albi can s was obtained from the American Type Culture Collection (ATCC 10231) and cultured in YPD medium (Current Protocols in Molecular Biology, Vol. 2, 13, Sup. 19 (1989)) at 30 ° C with vigorous agitation and aeration. All procedures were carried out at 4 ° C using 18 liter cultures. The cells were harvested by centrifugation (5000 rpm, 10 min., Sorval H6000 rotor), washed once with approximately 1 liter of ice cold deionized water and re-assembled in a pellet as above. The cell pellet (200-300 g wet weight) was completely resuspended in one volume of Shock Absorber A (50 mM Tris-HCl, pH 7. 9.4 ° C, 10% glycerol, lmM EDTA, 5 mM MgCl2, and protease inhibitor) with a content of 300 mM (NH () 2S0 (equivalent to the packed volume of cells (determined by weight, assuming a density of 1 g / mol of cells.) The cells that were resuspended were processed immediately as described below, or frozen by manipulating them with pipette in liquid N-liquid and storing them at -80 ° C. Frozen cells were thawed on ice before continuing, followed by the addition of NP-40 to a final concentration of 0.1%, the cells were broken by grinding with 1 ml of acid-washed glass beads / ml of cell suspension (Sigma, 400-625 μM ) using 12 irruptions of 30 seconds each, in an apparatus for the rupture by the shock with beads (BioSpec) The glass beads were allowed to stand and the supernatant was centrifuged at 30,000 xg for 40 minutes. iJíSO-i solid, slowly, until a concentrated Finally, 0.4 g / ml of supernatant was added and the resulting precipitate was pelletized by centrifugation at 100,000 x g for 30 minutes. The pellet was resuspended with a volume of Shock A sufficient to produce a conductivity equivalent to that of Shock Absorber A with a content of 75 iaM of (NH4) 2S04. After centrifugation of the resuspension, at 10,000 xg for 10 minutes, this supernatant (ca. 1-1.5 mg of protein / ml) was loaded onto a 300 ml column, DE-52 'DEAE-cellulose, equilibrated with the Shock absorber A with a content of 75 mM of (NH4) 2S04. After washing with 5 volumes of the column, of Shock absorber A with 75 mM of (NH4) 2S0, and 5 volumes of column, of Shock absorber A with 0.15 M of (NH4) 2S04, RNA polymerase II was eluted with 5 volumes of column, of Shock absorber A with 0.4 M of (NH4) 2S04. Fractions containing the value were collected maximum protein, determined by the absorbance at 280 nm and removed. The fraction removed was dialyzed against Buffer A containing 20% glycerol, for 3 hours, at 4 ° C. The eluate of 0.4 M of (NH4) 2S04, from the DEAE-cellulose (261 mg protein, 290 ml) was diluted with enough Buffer A to decrease the conductivity to the equivalent of Buffer A containing 0.15 M (NH4) 2S04, centrifuged at 10,000 xg, for 10 minutes and the supernatant HE loaded with a flow of 30 ml / h over a column of ml of DEAE-cellulose, equilibrated with Shock Absorber A containing 0.15 M (NH) 2S04, the column was developed with a linear gradient of 200 ml, 0.15-0.4 M (NH4) 2S04 in Shock Absorber A, with a flow of 45 ml / h. Fractions of the single peak or maximum value of RNA polymerase activity, sensitive to the anitine, were removed by eluting about 0.22 M (NH4) 2S04 (21.1 mg of protein, 45 ml) and loaded directly onto a column of 5 ml of Heparin agarose, equilibrated with the Shock A which contained 0. M of (NH4) 2S04. The column was washed with 3 volumes of column, of Shock Absorber A containing 0.2 M of (NH4) 2S04, and developed with a linear gradient of 80 ml of 0.2-0.6 M (NH4) 2S04 in Shock Absorber A. The active fractions, which eluted at approximately 0.42 M of (NH4) 2S04, were removed (2.0 mg of protein, 15 ml), frozen in 300 μl aliquots, in liquid N2, and stored at -80 ° C, in where the activity was stable for at least 6 months. The purification of the initiation factors of the proteins, used in the assay, is carried out by standard methods known in the art (for example, chromatography in phosphocellulose followed by gel filtration), as described in (Nature 346, 387-390 (1990)). For the selective classification of the transcription inhibition mediated by the TFIIB of Candida albicans, a transcription assay is reconstituted, using TFIIB of Candida albicans, recombinant. The TFIIB of Candida albícans is expressed there and purified from E. coli, as described in Burato ski, 1993, Proc. Nati Acad. Sci: 90: 5633. Plasmid DNA, supercoiled, is purified which contains the CYC1 promoter bound to the G-lower cartridge described by Lue et al. (Science 246, 661-664 (1989)), through standard methods for the purification of supercoiled circular DNA (Current Protocols in Molecular Biology, Vol. 2, 13, Sup. 19 (1989)). 10 to 100 ng of TFIIB of Candida albicans, 10 to 100 ng of TBP of Candida albicans, 10 to 100 ng of RNA polymerase II of Candida albicans, and 1 μg of plasmid DNA, are added to 50 μl of mixtures of reaction containing 50 mM HEPES, pH 7.5, 10% glycerol, 90 mM potassium glutamate, 0.75% polyethylene glycol (molecular weight 3350), 10 M magnesium acetate, 5 M EGTA, 5 mM DTT, 0.4 mM ATP, 0.4 mM CTP, μM [a-32P] UTP, 0.2 M of 3 '-O-methyl-GTP, and containing or lacking a candidate inhibitory molecule. The reactions are incubated at 30 ° C for a time of 30 to 60 minutes and RNA synthesis is detected as described below. b) Transcript RNA detection. The detection of the recently transcribed RNA is achieved through standard methods (Current Protocols in Molecular Biology, Vol. 1, 4.10, Sup. 24 (1989)). As an example, RNA synthesis can be detected as the incorporation of a radioactively or fluorescently labeled nucleotide, into higher molecular weight RNA products, determined by one of the following methods: 1) acid insoluble labeled material, quantified to by the appropriate method (eg, scintillation counting for dioactive precursors, fluorometry for fluorescent precursors); 2) labeled reaction product that hybridizes to oligonucleotides complementary to the correctly initiated transcript (ie, northern blot analysis); 3) the presence of a band marked with the appropriate mobility detected by autoradiography, on gels for electrophoresis based on denaturing polyacrylamide: 4) any other method that discriminates polynucleotide mononucleotides, wherein the polynucleotides are the product of the desired RNA. Those methods may use one or more well-known molecular biology techniques (Current Protocols in Molecular Biology, Vol. 2, 13, Sup. 19 (1989)), for example; UV analysis; affinity systems (e.g., affinity chromatography, nitrocellulose filtration, tretinoin, biosynthetic systems, immunoaffinity) (Current Protocols in Molecular Biology, Vol. 2, 13, Sup. 19 (1989)); and high resolution liquid chromatography. The inclusion of an inhibitory molecule that interferes with the biological activity of the TFIIB of Candi gives albi cans, inhibits transcription. In this assay the inhibition is measured as a reduction in the amount of the mRNA transcript produced relative to the amount of the transcript of mRNA produced in the absence of the inhibitor (the positive control) A decrease in the amount of the mRNA transcript is indicative of an inhibitor.

Effective levels of inhibition of the mRNA transcript are described below. " EXAMPLE 2 Selective Classification of the Inhibition of the Formation of the DNA-Protelna Complex A DNA-protein binding assay, consisting of the minimum components necessary to allow the association of the TFIIB of Candida albicans-RON to occur, can be used to classify the inhibition of the formation of the DNA-TBP-TFI IB complex of Candida albicans, during the inhibition of transcription. The components of that assay include: a) a DNA standard, b) TFIIB of Candida albícans, recombinant, c) preferably of Candida albicans,? _ Optionally d) an inhibitor of TFIIB of Candida albí cans, candidate. The inclusion of an inhibitory molecule that interferes with the interaction between the TFIIB_of Candida albicans and the DNA standard, inhibits the initiation of transcription. The inhibitor can interact directly with the TFIIB protein of the Can not give the bi can s, and / or can interact with the T3P and / or with the DNA pattern at the TFIIB / TBP binding site. In this assay the inhibition is measured as a reduction in the amount 5 of the DNA-TBP-TFII complex produced relative to the amount of the DNA-TBP-TFI IB complex produced in the absence of the inhibitor (the positive control). A decrease in the amount of the DNA-TBP-TFIIB complex is indicative of an inhibitor. The Determination of effective levels of DNA-TBP-TFIIB inhibition is described below. A DNA binding assay is constructed as follows. An amount of 10 to 100 ng of TFIIB of Candi da albi can s, expressed and purified in E Coli as described above, is incubated with 0.5 ng of labeled oligonucleotide (eg, radioactively or fluorescently labeled) which contains a TATA element such as that described by Buratowski et al. (Cell 56, 549-561 (1989) and an amount of 10 to 100 ng of Candi TBP given to lbi can s, in reactions containing 10 to 20 mM HEPES (or equivalent), pH 7.5-8.0, 5 mM MgCl2 , 12% glycerol, 10 mM dithiothreitol (DTT), 100 μg / ml BSA, 5-20 μg / ml poly (dG- 5 dG): (dG-dC) and a candidate inhibitor of the formation of the complex. The reactions are incubated at 30 ° C for a time of 30 to 60 minutes. The formation of the DNA-TBP-TFIIB complex can be detected as the retention of labeled DNA (labeling is detected through an appropriate methodology such as scintillation counting, for radiolabeled DNA, or fluorometry for fluorescently labeled DNA) using methods of known affinity for the immobilization of proteins (for example, biotin / streptavidin, nitrocellulose filtration, affinity chromatography, immunoaffinity). The non-retention of the labeled DNA, due to the failure in the formation of the TFIIB-TBP-DNA complex of Candi da albi can s, is indicative of an effective inhibitor. The formation of the complex can also be detected as the retention of the TFIIB from Candi gives bi-labeled drugs (for example, radioactively, fluorescently!) Using well-known methods to immobilize DNA.The non-retention of TFIIB from Candi gives labeled proteins, due to the failure in the formation of the TFIIB-TBP-DNA complex. of the Can di da to bi c an s, is indicative of an effective inhibitor The two preceding methods are suitable for applications of ? analysis of libraries with chemical compounds in large volumes, such as those commonly used in drug discovery. A third example for detecting the formation of the DNA / protein complex involves the detection of a shift in the electrophoretic mobility of the labeled DNA in 4% polyacrylamide gels containing 5% (v / v) glycerol, 25 mM Tris. , 100 mM of glycine, lmM of # 10 EDTA, 5 mM MgCl2, pH 8.3, in the presence of TFIIB and TBP of Candi da albí cans. The position of the labeled oligonucleotide is detected by appropriate methods (for example, autoradiography, for a radioactive oligonucleotide). The absence or The deviation of the expected displacement in mobility, due to the formation of the DNA-TFIIB complex of Candi da albi can s, is indicative of an effective inhibitor. Finally, other methods can be used to detect or separate "DNA-protein complexes, including UV cross-linking analysis, high-resolution liquid chromatography, phage display technology (US Patent No. 5,403,484.

Chimeric Protein Enlaging), and resonance of surface plasmon (Biacore, Pharmacia Biosensor, North America) as described below.

EXAMPLE 3 Selective Classification of the Inhibition of Protein-Protein Complex Formation - A protein-protein binding assay can be used that consists of the minimum components necessary to allow the binding of the TBP of Candida albicans-TFIIB from Candi to Ib i cans to selectively classify the inhibition of complex formation. TBP of Candida albicans-TFII of Candida albicans, during the initiation of transcription. The elements of "such an assay consist of: a) TFIIB of Candida albicans, recombinant, b) TBP, preferably a TBP of Candida albicans, recombinant, and optionally c) a candidate linker inhibitor." The inclusion of an inhibitory molecule that interferes with the interaction between the TBP of Candida albicans and the TFIIB of Candida alibi cans inhibits the initiation of transcription. The inhibitor can interact with the TBP protein or TFIIB of Candida albicans and thus induce a configurational change that prevents binding, or it can directly inhibit the interaction of the TFIIB and TBP proteins of Candida albicans. In this assay, the inhibition is measured as a reduction in the amount produced of the TBP-TFIIB complex of Candida albicans, relative to the amount produced of the TBP-TFIIB complex of Candida albicans in the absence of the inhibitor (the positive control). A decrease in the amount of the TFIIB-TBP complex is indicative of an inhibitor. The determination of the effective inhibition levels of the TBP-TFIIB binding of Candi da albi cans is described below. An assay for the formation of the TBP-TFIIB complex of Candida albicans is as follows. They are expressed, from 10 to 100 ng of TFIIB of Candida albicans and of 10 to 100 ng of TBP of Candida albicans, in E. Coli, and purified therein, as described above, and are added to reactions containing 10 to 20 M HEPES (or equivalent), pH 7.5-8.0, 5 M ? MgCl2, 12 glycerol, 10 mM "of di tiodide 1 (DTT) 100 μg / ml of BSA, and a candidate inhibitor, then incubate the mixture at 30 ° C for 30 to 60 minutes. 5 The formation of a complex comprising TBP of Candida albicans and TFIIB of Candida albicans can be detected through the displacement of the electrophobic mobility of the TBP or radiolabeled TFIIB (for example, # 10 radioactive or fluorescent) in polyacrylamide gels containing 5% (v / v) glycerol, 25 mM Tris, 100 M glycerin, 1 mM EDTA, 5 mM MgCl2, pH 8.3, in the presence of the companion not marked. The position of the labeled partner is detected by appropriate methods (e.g., t autoradiography for the radioactive oligonucleotide). The absence or derivation of the expected displacement of mobility due to The formation of the TFIIB-TBP complex of Candida albicans is indicative of an effective inhibitor. The formation of a complex comprising TBP from Candida albicans and TFIIB-from Candida albicans can be detected as the retained TBP, using methods of known affinities for the immobilization of the TFIIB protein of Can di da albi cans (for example, bio tine / is trept avidin, nitrocellulose filtration, affinity chromatography, immunoaffinity). Failure in the formation of the TFIIB-TBP complex of Candi da lbi can s is indicative of inhibition, and is indicated by the non-retention of labeled TBP. Alternatively, the immobilized element can be the TBP of the Can di da a bi can s and the companion marked the TFIIB of the Ca n di da a la b can s. In the previous example, a stronger signal can be conferred in the presence of both the TBP and the TFIIB and, in addition, a DNA standard containing a TATA element. The complex is then quantified by author adiography, phosphorus imaging technology, or counting of signals for radioactively labeled factors, fluorometry for fluorescently labeled factors, luminometry for factors labeled with ligands that are detected using chemiluminescent sounding methodologies. or phosphorescent, or other similar detection methods, or marked materials as described above, which are standards in the art. Other methods can be used to detect or separate protein-protein complexes, which include UV cross-linking analysis, high-resolution liquid chromatography, phage display technology, and surface plasmon resonance, as described at the moment .

EXAMPLE 4 Assay for the Formation of the TBP-TFIIB-RNA Polymerase I I-DNA Complex It is known that the formation of the TBP complex, TFIIB, RNA polymerase II, DNA, is notably stimulated by the addition of another factor, the TFIIF. The previous data indicate that the TFIIF of S. I believe that it can work in related species as distantly as the Crassomyces pombe and humans, suggesting strongly that this factor can functionally replace its canon with a counterpart. Therefore this factor is purified from 5. cerevisiae through published methods (Sayre, 1992, J. Biol. Chem. 267: 23383) and used to reconstitute the formation of a complex containing TBP, TFIIB, and RNA polymerase II, from Candi gives 5 to lb, and a promoter containing DNA such as that described for the reconstitution of the TFI IB-TBP-DNA complex. The formation of the complex is carried out in reactions that contain, for example, to 100 ng of TBP of the Ca n d to the bi cans, from 10 to 100 ng of TFIIB of the Candi da albi can s, from 10 to 100 ng of RNA polymerase II of Candi da albi can s, from 10 to 100 ng of the TFIIF of 5. c ere vi yes ae, 0.5 g of oligonucleotide containing the TATA element of double strand (the same one used for the TFI IB-TBP-DNA complex assay), 10 to 20 mM HEPES (or equivalent), pH 7.5 to 8.0, 5 mM MgCl2, 12% glycerol, 10 mM dithiothreitol (DTT), 100 μg / ml BSA, 5 to 20 μg / ml poly (dG-dC); (dG-dC) and compound (s) to be analyzed with respect to their inhibitory activity. After incubation at 30 ° C for 30 to 60 minutes, the complexes are detected by the methods described above for the TBP-TFI IB-DNA complex. The complex of TBP-TFIIB-RNA polymerase I I-DNA has less electrophoretic mobility than the TBP-TFI IB-DNA complex identified through the use of the electrophoretic method. In addition, the formation of the complex can be detected as a retention, dependent on TBP and TFIIB, of the activity of RNA polymerase II (measured by the incorporation of marked tyridic core precursors, to the acid insoluble product, using the assay for the activity of the RNA polymerase described in the protocol for the purification of the RNA polymerase II mentioned above) in a matrix with TATA element bound and containing DNA. The IC50 of the inhibitory compounds will be determined by titration in reconstituted reactions, as described above. The IC_0 of these compounds against reconstituted reactions with TBP, TFIIB and human polymeric RNA II, will also be determined by the same method. Human RNA polymerase II and TFIIB are purified as previously described (Flores et al. ., 1990, J. Biol. Chem. 265: 5629-5634, Reinberg et al., J. Biol. Chem 262: 3310-3321). The compounds whose IC5o, against reactions that contain factors of the Candi da albí cans, is less than or equal to its ICB0 against reactions reconstituted with human factors, will be analyzed with respect to its ability to inhibit the growth of Can di da a l b ca n s, as will be described later.

EXAMPLE 5 Selective Classification of the Inhibitor by Phage display In addition to the standard techniques mentioned above, other technologies can be used for molecular identification, in the identification of inhibitory molecules. One of these technologies is the phage display technology (US Patent No. 5,403,484, Viruses Expressing Chimeric Binding Proteins). The phage display allows the identification of a binding protein against a selected target. The phage display is a selective molecular classification protocol that uses recombinant bacteriophages. The technology involves transforming bateriophages with a gene that encodes an appropriate ligand (in this case, a candidate inhibitor) capable of binding to the target molecule of interest. For the purposes of this description, the target molecule may be the TFIIB of Can di da al bi c an s, or a DNA-protein or protein-protein complex formed using TBP and / or TFIIB, as described herein. The transformed bacteriophage (which is preferably attached to a solid support) expresses the candidate inhibitor and displays it on its phage coat. The cells or viruses that carry the candidate inhibitor, which recognize the target molecule, "are isolated and amplified, then the successful inhibitors are characterized." Phage display technology has advantages over standard technologies of selective affinity ligand classification. The phage surface exhibits the microprotein ligand in a three-dimensional arrangement, which more closely resembles its natural configuration.This allows for a more specific and more affinity binding for selective classification purposes.

EXAMPLE 6 Analysis of Biospecific Interaction A second, relatively new, selective classification technology that can be applied to selective inhibitor classi fi cation assays is the biospecific interaction analysis (BIAcore, Pharmacia Biosensor AB, Uppsala, Sweden). This technology is described in detail by Jonsson et al. (Biotechniques 11: 5, 620-627 (1991)). The biospecific interaction analysis uses surface plasm resonance (SPR) to inspect the adsorption of biomolecular complexes on a chip of detector integrated circuits. The SPR measures changes in the refractive index of a polarized light directed at the surface of the detector chip. Specific ligands (ie, -the candidate inhibitors) capable of binding to the target molecule of interest (ie, the TFIIB of the Can di gives the bi ca nso a protein-protein complex "or of protein-DNA, which contains TFIIB) they are immobilized in the detector chip. In the presence of the white molecule, binding specific to the immobilized ligand is presented. The incipient ligand-white molecule complex causes a change in the refractive index of the polarized light and is detected by a diode array. The analysis of biospecific interaction provides the advantages of; 1) allow studies without the need for marking, of the formation of a molecular complex; 2) study the molecular interactions in real time when the test is passed through the detector chip; 3) detect surface concentrations below 10 pg / mm2; detecting the interactions between two or more molecules; and 4) is fully automated (Biotechniques 11: 5, 620-627 (1991)).

EXAMPLE 7 Large Volume Selective Classification of Potential Inhibitors In accordance with the invention it is contemplated that the selective classification methods described herein encompass the selective classification of multiple samples simultaneously, which is also referred to herein as selective classification "in large volumes". For example, selective sorting in large volumes can selectively range from several hundred to several thousand candidate inhibitors in a single test. An example of a large volume selective sorting test according to the invention is as follows. A protein fusion protein A 8pA) -TFIIB from Can di da a bi can s is generated by inserting the TFIIB coding sequence into the downstream structure of the pA coding sequence of plasmipRIT2T (Pharmacia Biotech). The fusion construct is induced and the resulting recombinant protein is extracted and purified according to the conditions recommended by the manufacturer. This procedure can also be carried out for the preparation of a fusion protein of pA-TBP from the Candi gives to the bi cans except that the downstream coding sequence is that of the TBP protein; all the other steps They would remain as such. A 2 microliter Dynatech Microlite plate, or high capacity equivalent plate for protein binding, is coated with 1 μg / well of 300 μl human IgG of 3.33 μg / ml human IgG (Sigma) in coating buffer ( 0.2 M sodium carbonate, pH 9.4) in the well, for a period of 4 to 12 hours, at a temperature of 4 ° C. The buffer is then decanted for coatings and the wells are washed five times with 300 μl of PBS. 300 μl of blocking buffer (SuperBlo kMR blocking buffer, Pierce) containing pA-TFIIB or pA-TFIIB is added at a rate of 3.33 μg / ml, and the plate is incubated for 4 hours or more at 4'C. -the plates can be stored in this form at 4 ° C until ready for use- When ready the plates are washed five times with 300μl of PBS.The test compound, at a final concentration of 20 to 200 μM and TFIIB or labeled TBP (ie, non-fusion protein), provided that they are not added during the coating step, and 10 to 1000 fmol of DNA containing a TATA sequence, are suspended in HEG buffer which contains 200 μg / ml of BSA in a total volume of 150 μl and added, and the reaction is incubated at room temperature with gentle agitation for 60 minutes. Then the plate is washed five times with PBS using a Dynatech plate washer or equivalent device. The labeled and bound protein is quantified by adding 250 μl of Microscint (Packard) per well and counting in a scintillation spectrophotometer, compatible with the microtitre plate.As an alternative, the fusion of protein A and protein The second non-fusion protein can be incubated in the presence of the test compound in polypropylene microtiter plates under the same buffer and under the same incubation conditions described above, then the reaction mixture is transferred to the wells. of a microtiter plate coated with human IgG (which is prepared as described above, and stored in the blocking buffer and washed five times with 300 μl of PBS immediately before use) and incubated for 60 minutes At room temperature with gentle agitation, the retention of the radioactive protein 9 quantify as above. The interaction of TBP and TFIIB, which is measured as the retention of radioactivity in the plaque, is dependent on the human IgG that covers the plaque, and on the TBP or TFIIB of the Candi da albi cans wild type, one of the which must be merged into the pA. Candidate inhibitors or extracts that inhibit retention ^ | of radioactivity by more than 30% - they are identified, and the inhibitory activity is further purified if necessary. "The inhibitors are then identified, as described above, with respect to their ability to inhibit TFIIB-dependent transcription. of the Can di da aljbi ca ns, in an in vitro transcription system such as the one JB described here, and can also be analyzed with respect to its capacity to inhibit the growth of the Candi da bi can s . Other systems of fusion or modified proteins, which are contemplated, include, but are not limited to, glutathione-S-transferase, the maltose binding protein, the hemagglutinin of the influenza; the FLAGM and mergers of the hexahist idina to the TBP of the Candi gives to the bi can s_ to the TFIIB of the Can di da albi cans, which are prepared, expressed and purified through published methods, or TBP or TFIIB of the Can be given to the biomes treated with biotin, which are prepared using reactive biotin precursors that are commercially available. The purified or modified fusion protein is immobilized on a microtitre plate containing the appropriate ligand for each fusion protein (eg, glutathione, amylose, CA157 antibody, etc., respectively), the assay is carried out and the results are evaluated essentially in the same manner as described above.

EXAMPLE Candidate Inhibitors A "candidate inhibitor", as used herein, is any compound with a potential to inhibit the initiation of transcription mediated by the TFIIB of the Candi da canibi, or to inhibit the formation of a complex. A candidate inhibitor is analyzed in a concentration range that depends on the molecular weight of the molecule and the type of assay. For example, for the inhibition of protein / protein or protein / DNA complex formation, small molecules (as defined below) can be analyzed in a range of concentrations of lpg-100 μg / ml, preferably to approximately 100 pg - 20 μg / ml; Large molecules, for example, peptides, can be analyzed in the range of 10 ng-100 μg / ml, preferably 100 ng-10 μg / ml. The inhibitors of growth or viability of Candi gives the bi can s target in the novel transcription factor described here, the TFIIB, or they can target a protein or nucleic acid that interacts with the novel transcription factor, to preventing the "natural biological interaction that occurs i vi and v leads to the initiation of transcription in Candida." Thus, an inhibitor identified as described herein will possess two properties: 1) at a certain concentration it will inhibit the growth or < ?, viability of Can di da a lbi cans; and 2) at the same concentration, will not significantly affect the growth of the cells of a mammal, particularly of humans. The inhibitory candidates will include peptide and polypeptide inhibitors having an amino acid sequence based on the sequences of the novel TFIIB disclosed herein. For example, a fragment of TFIIB can act as a competitive inhibitor with respect to the binding of TFIIB to other proteins involved in transcription of Candida, for example, RNA polymerase II, TBP, or with respect to binding of the complex of transcription to the DNA template. One of those candidate fragments is a fragment of TFIIB JjÉ that contains a classic motif (sequence) of zinc finger. This fragment spans a region of 35 amino acids defined by the residues 22-46 of the TFIIB sequence. The candidate inhibitory compounds of large libraries of synthetic O-natural compounds can be selectively classified. Many means are commonly used for "the direct and random synthesis of saccharides, peptides, and compounds based on nucleic acids. The libraries of synthetic compounds are available commercially in a number of companies which include the Maybridge Chemical Co. (Trevillet, Corn all, UK), Comgenex (Princeton, NJ), Brandon Associates (Merri ack, NH), and Microsource (New Milford, CT). A rare library is available at Aldrich (Milwaukee, Wl). Combinatorial libraries are available and can be prepared. Alternatively, libraries of natural compounds, in the form of extracts of bacteria, fungi, plants and animals, are available, for example, from Pan Laboratories (Bothell, WA) or MycoSearch (NC), or they can be produced easily. Additionally, libraries and compounds, produced naturally and synthetically, are easily modified through chemical, physical and biochemical, conventional means. Useful compounds can be found in numerous chemical classes, although typically they are "organic compounds, and preferably small organic compounds." Small organic compounds have a higher molecular weight of 50 but less than about 2,500 daltons, preferably less than about 750, more preferably less than about 350 daltons. Exemplary classes include compounds, compounds, peptides, saccharides, steroids, and the like. The compounds can be modified to increase their efficacy, stability, pharmaceutical compatibility, and similar properties. The structural identification of an agent can be used to selectively identify, generate, or classify additional agents. For example, where peptide agents are identified, they can be modified in a variety of ways to increase their stability, such as the use of an unnatural amino acid, such as a D-amino acid, particularly D-alanine, functions 1 initiating the amino or carboxylic terminus, for example for the amino group, the acylation or the alkylation, and for the carboxyl group, the esterification or the amidification, or the like. Other methods of stabilization may include encapsulation, for example, in liposomes, etc.

EXAMPLE 9 - Measurement of effective inhibition The amount of inhibition performed by a candidate inhibitor is quantified using the following formula which describes reactions reconstituted with a radioactively labeled portion: Positive - CPMMußßtra) Percent Inhibition = X 100 (CPM Positive Control) where CPMcontroi p siti is the average of the cpm in complexes or in RNA molecules formed in reactions lacking the candidate inhibitor, and CPMMu «st -._- is the cpm in complexes formed in reactions that contain either the TFIIB of Candida albícans or human TFIIB (expressed in E. coli and purified therefrom, using the existing recombinant clones (Peterson et al., Science 248, 1625-1630, 1990; Kao et al., Science 248, 1646- 1650, 1990; Hoff an, "et al., Nature 346, 387-390, 1990, and analyzed as described above) and its IC50, with respect to human TFIIB and Candida albicans, determined from graphs of the concentration of the compound vs. % inhibition. ICBo is defined as the concentration that results in a 50% inhibition. Candidate inhibitors for which the IC5o, against reactions containing the TFIIB of Candida albicans, is less than or equal to 1/5 of the IC_o or against reactions containing human TFIIB, are further analyzed for their ability to inhibit the growth of Candida albicans in crops, as described later.

EXAMPLE 10 Measurement of growth inhibition -of Candida albícans in crops- Once the inhibitor is identified in one or more of the binding or transcription assays described herein, it may be desired to determine the effect of the inhibitor on the growth and / or viability of Candida albicans in crops. The ability of a candidate inhibitor to inhibit the growth of the cells of Ca biology in cultures is analyzed, as follows. Methods for performing tests of growth inhibition in cultures are well known in the art. One of these procedures is based on the NCCLS method M27P (The National Committee for Clinical Laboratory Standards, Reference Method for the Test of Antifungal Susceptibility, Yeast, with Dilution of the Culture Broth, proposed standard, 1992), as follows. Serial dilutions (two to three times steps starting at a maximum concentration of 100 to 200 μg / ml) of the candidate inhibitor are prepared using RPMI-1640 medium as diluent and an aliquot of 100 μl of each dilution is added to each of the polystyrene microtiter plates, "of 96 wells- Five colonies of Can di da the bi can s are suspended, taken from a plate with Dextrose Sabouraud Agar inoculated from 14 to 20 hours previously with the Can di da strain. To the analysis bios (Catalog Number 10231 of the Yeast Catalog of the American Type Culture Collection), they are suspended with RAMI-1640 media in such a way that the density of cells is from 10,000 to 30,000 cells / ml. 100 μl of the cell suspension is added to each of the wells of a 96-well microtiter plate containing the diluted candidate inhibitor and the control medium. The cultures are mixed with shaking and incubated at 35 ° C for 48 hours without agitation, after which cell growth is verified by visual inspection by the formation of turbidity - and / or mycelial colonies. The minimum concentration of the candidate inhibitor at which no cell growth is detected by this method is defined as the minimum inhibitory concentration (MIC) for that compound. Examples of MICs for known antifungal compounds obtained using this technique are 0.125 to 0.5 μg / ml for fluconazole and 0.25 to 1.0 μg / ml for amphotericin B (The National Committee for Clinical Laboratories and Standards, Reference for the Test of Susceptibility Antifúngica, of Yeasts, with Dilution of the Broth of Cultivation, proposed norm, 1992). An inhibitor identified by the methods described herein will have a MIC that is equivalent to or less than the MICs for fluconazole or for the amphotericin B.

EXAMPLE 11 Selective counter-classification of the Inhibition of Transcription, Using Human TFIIB A compound identified as an inhibitor of Can di da lbi can s, in accordance with one or more of the assays described herein may be further analyzed in order to determine its effect on the host organism. of antifungal compounds useful for human therapy, it is desirable that these compounds act as effective agents in inhibiting the viability of the fungal pathogen and that they do not significantly inhibit human cellular systems, specifically, the inhibitors of Candi give the bi can be identified in any of the assays described above, can be subjected to a reverse selective classification for the inhibition of human TFIIB.The recombinant human TFIIB can be obtained from existing sources and be purified by known methods (for example, see Peterson et al., Kao et al., and Hoffman et al., supra) and can be contacted with the candidate inhibitor in assays such as those described above using a human system. The effectiveness of an inhibitor of Can di da albi can TFIIB, as a human therapeutic agent, is determined as one that exhibits a low level of inhibition against human TFIIB, in relation to the level of inhibition with respect to the TFIIB of Ca ndi da to the bi can s. For example, it is preferred that the amount of inhibition caused by a given human TFIIB inhibitor, in a human system, is not greater than 20% with respect to the amount of inhibition of TFIIB of Can di da albi can s, in a Can di da system, when analyzed in any of the tests described above.

Dosage and Pharmaceutical Formulations For therapeutic uses, the inhibitors identified as described herein may be administered as a formulation pharmaceutically compatible / biologically compatible. For example, in the form of a cream, ointment, lotion or sprayer, for topical use, or in the form of a physiological solution, such as a saline solution, for internal administration. The amount of inhibitor administered will be determined according to the degree of pathogenic infection and depending on whether the infection is systemic or localized, and will typically be in the range of from about 1 μg to 100 mg / kg of body weight. Where the inhibitor is a peptide or a polypeptide, it will be administered in the range of from about 100 to 500 μg / ml per dose. In accordance with the invention, a single dose of the inhibitor or multiple doses thereof is contemplated daily, weekly or intently. The route of administration will be chosen by the physician, and may be topical, oral, transdermal, nasal, rectal, intravenous, intramuscular, or subcutaneous.

Deposit according to the Budapest Treaty On September 15, 1995, A.T.C.C., Rockville, MD, was deposited at an international deposit center, under accession number 69900, an E. c or l transformed with a plasmid that contains the gene that codes TBP of Can di da a lbi ca n s. On September 15, 1995, the A.T.C.C., Rockville, MD, was deposited at an international depository center, under accession number 69899, an E. col i transformed with a plasmid containing the gene encoding the TFIIB of Can di da al bib can s. The Numbers 69900 and 69889 of the A.T.C.C. they will be available to the public upon granting of a patent describing the access numbers together with the invention described herein. The deposits were made under the Budapest Treaty, and will be available after the life that may be required for the patent for which the deposit was made, and will be maintained for a period of at least 30 years from the date of the deposit. deposit and at least 5 years after the ATCC Receive the most recent request to supply a sample of the deposit. You must it being understood that the availability of these deposits does not constitute a license to carry out the invention in question in derogation of the patent rights granted for the invention in question by governmental action.

OTHER MODALITIES The preceding examples demonstrate the experiments carried out and contemplated by the inventors herein, when carrying out and carrying out the invention, it is believed that these experiments include a description of the techniques that serve both to assess the technique of carrying out the invention as to demonstrate its utility Those skilled in the art will appreciate that the techniques and modalities described herein are preferred embodiments only that, in general, numerous equivalent methods and techniques can be used to achieve the same result. previously in the present are expressly incorporated therein, as reference, to the extent of what they describe, and what is disclosed provides a basis for enabling the compositions and / or methods that may be important to carry out one or more embodiments of the present invention.

It is noted that in relation to this date, the best method known by the applicant to carry out said invention, "is that which is clear from the present description of the invention." Having described the invention as above, it is claimed As property what is contained in the following: (i) APPLICANT: SCRIPTGEN PHARMACEUTICALS, INC. (Ü) TITLE OF THE INVENTION: NEW TRANSFER FACTOR TFIIB OF CANDIDA ALBICANS ... (iii) SEQUENCE NUMBER: 4 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: ARBY £ ARBY P.C. (B) STREET: 805 Third Avenue (C) CITY: New York (D) STATE: New York (E) COUNTRY: United States of America (F) ZIP: 10022-7513 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: Compatible with IBM (C) OPERATING SYSTEM: DOS (D) SOFTWARE: FastSEQ Version 1.5 (vi) COMMON DATA OF THE APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF PRESENTATION: (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: 08/625, 377 (B) DATE OF SUBMISSION: 01-APRIL-1996 (viii) INFORMATION OF THE LAWYER / MANDATORY: (A) NAME: S. PETER LUDKING, ESQ. (B) REGISTRATION NUMBER: 35,351 (C) REFERENCE / FILE NUMBER: 0342 / 2C488-WO (ix) INFORMATION FOR TELECOMMUNICATION: (A) TELEPHONE: (212) 527-7700 (B) TELEFAX: (212) 753-6237 (C) TELEX: (2) INFORMATION FOR SEC ID No. 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 219 amino acids (B) TYPE: amino acid (C) HEBRAS: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (Üi) HYPOTHETICAL: NO (iv) REVERSE SENSE: NO (v) TYPE OF FRAGMENT: internal (vi) ORIGINAL SOURCE: (xi) DESCRIPTION OF THE SEQUENCE: SEC ID No. I: 11 = t-lu Asp Thr Thr 5 30 Asp Thr Leu sln Asn 45 He Leu Lys Thr lie Ala Arg phe Ala Ala S5 80 Val Leu lie phe Ala 95 Ser Asp Ser Lys no Leu Leu sly Phe Asn 125 Wing Gly Ser Thr Asp Val Ala Hls Gly Thr 145 160 Phe lie Tyr Arg Met 175 Val Gly Lys lie Val 25 190 Leu Wing Phe Glu Ser 205 * - • £ - He (2) INFORMATION FOR SEC ID No. 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 344 amino acids 3S (B) TYPE: amino acids (C) HEBRAS: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide 4 0 (lü) HYPOTHETICAL: NO (IV) REVERSE SENSE: NO (v) TI PO DE FRAGMENT: internal (vi) ORIGINAL SOURCE: 4 5 (xi) DESCRIPTION OF THE SEQUENCE: SEC ID No. 2: Met Ser Pro Ser Ti-r Ser Thr Ala Val Gln Glu Tyr lie Gly Pro Asn 1 S 10 15 50 Leu Asn Val Thr Leu T r Cys Pro Giu Cys Lys lie Phe Pro Pro Asp 20 25 30 Leu Val Glu Arg Pl -e --ex Glu Gly Asp He Val ands Oly Ser Cys Gly 35 40 45 Leu Val Leu Ser Asp Arg Val Val Asp Thr Arg Ser Glu Trp Arg Thr 55 50 55 SO Phe Ser As Asp Asp Gln As Asp Asp Asp Asp Arg val aly Asp 55 70 75 80 Wing Gly Asn Pro Leu Leu Asp Thr Glu Asp Leu Ser Thr MeClia Ser 85 90 95 I0 Tyr Ala Pro Asp Ser Thr Lys Ala sly Arg alu Leu Ser Arg Ala Gln loo ios lio Ser Lys Ser Leu Val Asp Lys Lys Asp Asn Ala Leu Ala Ala Ala Tyr 115 120 123 He Lys He Ser Wave Mee Cys Asp Oly Tyr Gl-- Leu Pro Lys He Val 65 130 135 140 Ser Asp Gly Wing L s Glu Val Tyr Lys Mae Val Tyr A = p Glu Lys Pro 145 150 155 ISO ys a sn ue rp a 180 185 190 5 Lys Thr f - n Val Pro Arg Lys slu He Gly Lys Val he Lys He Mee 135 200 205 Asp Lys He He Arg olu Lys Asn Ala Wing Asn Pro Asn Wing Ala Tyr 210 215 220 Tyr aly aln Asp Ser He aln Thr Thr sln Thr Ser Wing Glu Asp Leu 10 225 230 235 240 He Arg Arg Phe Cys Ser His Leu Gly Val Asn Thr sln Val Thr Asn 245 250 255 aly Wing Glu Tyr He Wing Arg Arg Cys Lys slu Val sly Val Leu Wing 250 265 270 15 sly Arg Ser Pro Thr Thr He Ala Wing Thr Val He Tyr Mee Wing Ser 275 280 285 Leu Val Phe Gly Phe Asp Read Pro Pro Ser Lys He Ser Asp Lvs Thr 290 295 300 sly Val Ser Asp Oly Thr He Lys T r Ser Tyr Lys Tyr Mee Tyr alu 20 305 310 315 320 Glu Lys alu aln Leu He- Asp Pro Ser Trp He Olu Ser Cly Lvs Val 325 330 335 Lys Leu Glu Ly3 He Pro Lys Asn 340 25 (2) INFORMATION FOR SEC ID No. 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 657 base pairs 30 (B) TYPE: nucleic acid (C) HEBRAS: unique (D) TOPOLOGY: linear -_ (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) REVERSE SENSE : NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: 40 (xi) SEQUENCE DESCRIPTION: SEQ ID No.3: ATGAAOTCAA TAGAGGAAGA TGAAAAAAAT AAAGCCGAA5 ATTTGGATAT TATAAAAAAC SO aAAaATATTG ATGAACCTAA ACAAGAAsAT ACCACTGATA GTAATGGTGG TGGAGGTATT 120 S GGTATAOTOC CCACATTACA AAATATTCTT GCTACGGTGA ATCTTGATTG TCGACTTOAT 130 AAAACAATTG CTTTACATGC TAGAAATGCC OAATATAATC CAAAACGTTt TGCTGCGsTs 240 ATTATGAOAA TTAsAaATCC AAAAACTACO OCATTAATCT TTGCTTCGOa GAAAATGGTT 300 aTGACTGGGG CTAAATCCGA AGACGATTCC AAGTTGGCTT CAAGAAAGTA TGCTAOAATC 350 ATTCAAAAGT TGGGGTTCAA TGCTAAATTT TGTGATTTTA AAATTCAAAA TATAGTGGGO 420 TCAACAGATG TTAAGTTTGC TATTAGATTA GAAGOCTTAO CTTTTGCTCA TOGTACTTTT 480 TCTTCATATG AACCAOAATT ATTTCCTGGa TTAATTTATA GAATOOTOAA ACCAAAAATT 540 GTTTTACTTA TATTTOTTTC TGaGAAAATT OTTTTGACsG sTaCCAAAAA OACAGAAGAA 600 ATTTATGATG CATTTOAACT OATTTATCCO OTTTTAAATO AATTTCaTAA AAATTGA 657 55 (2) INFORMATION FOR SEC ID No. 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1095 base pairs SO (B) TYPE: nucleic acid (C) HEBRAS: (D) TOPOLOGY: (V) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) DESCRIPTION OF THE SEQUENCE: SEC ID No. 4: TAAGCTTGTA TTACTAAGCA TATTATGTCG CCATCAACAT CTACGGCAGT ACAsGAGTAT 60 ATTGGACCAA ACTTGAATGT TACATTAACA TGTCCTGAGT GTAAGATATT TCCACCTOAT 120 TTGOTAGAsA aaTTCAGCGA AGGTGACATT GTCTGTGaCA GTTGTGGGCT AOTATtGAGT 180 aATCGTGTTG TGGATACGAG ATCAsAATGa AGAACTTTCA GTAACGATGA CCAAAATGGT 240 GATGATCCTT CTCGTGTTaa TGATGCAGOT AACCCTTTAT TAGACACAGA ssACTTGTCC 300 ACAATGATTT CTTATGCTCC TOATACTACC AAAGCAGGAA GAGAGTTAAG CCGAGCCCAA 360 TCTAAATCTC TAGTCGATAA AAAAOACAAT aCATTaGCTa CAGCATATAT CAAGATTTCT 420 CAAATGTGCG ATGGTTATCA ATTGCCTAAA ATAGTTCTGO ATGOaaCCAA COAAOTCTAC 480 AAAATsGTTT ATGACGAGAA ACCATTGCGA GGAAAATCAC AAGAGAGTAT CATsOCAsCT 540 TCTATCTTTA TTssTTsCAG AAAGGCCAAT GTTsCTCGTT CATTCAAAsA OR TATsGGCA so o AAGACTAATs TACCTCGTAA aGAAATTGGT AAAGTGTTCA AGATCATsGA CAAGATCATT 6S0 CGTGAAAAGA ATOCAGCCAA CCCTAATGCT OCATATTACG CTCAAGACAs CATTCAAACC 720 ACCCAAACTT CGGCCGAGGA TTTGATTAGA AGATTCTGTT CTCACTTGGG TG TAACACA 780 CAAGTTACAA ATGsTGCsaA ATACATAGCC AOAAaATGTA AaaAAGTCGG GGTTTTAaCA 840 GOTAGATCGC CAACTACAAT TaCTOCAACT GTAATTTACA TOGCTTCACT AGTGTTTGGA 900 TTTGACTTAC CTCCATCCAA aATATCTGAT AAAACTGGTO TCAGTGATGa TACTATCAAA 960 ACTTCATACA AGTACATGTA CGAGsAGAAA GAACAATTGA TTGATCCATC TTOGATAOAA 1020 AaTGGTAAAG TAAAATTGGA AAAAATACCA AAAAACTAAT ACAGCGGAGT CGCCACTGTT 1080 AATCCTITAC CCTCT 1095

Claims (15)

1. A recombinant nucleic acid, characterized in that it comprises an amino acid sequence encoding the transcription factor, TFIIB, of Candida albicans.

2. A vector, characterized in that it comprises a nucleic acid sequence - encoding the TFIIB of Candida albicans.

3. A transformed host cell, characterized in that it comprises a sequence of nucleic acids encoding the TFIIB of Candida albícans.

4. A recombinant polypeptide, characterized in that it comprises the TFIIB of Candida albícans.

5. A fragment of the TFIIB of Candida albi cans, characterized in that this fragment inhibits the biological activity of the TFIIB of Candi da albi cans at the start of transcription.

6. A fragment of the TF1IB of Candida albi cans, and this fragment is characterized because it prevents the growth of Candida albi cans.

7. A method for producing the TFIIB of Candida albicans ecombinant, characterized in that it comprises culturing the host cell according to claim 3, under 10 conditions sufficient to allow expression of the nucleic acid encoding the TFIIB of Candida albicans, and isolate the TFIIB from Candida albicans. 15

8. A method of selective classification to identify a growth inhibitor of Candida albicans, the method is characterized • ^^ because it comprises detecting the inhibition of transcription of the mRNA in a test or analysis of 20 in vitro transcription comprising a DNA template or template, RNA polymerase II, TFIIB of the recombinant Candida albicans, and a candidate inhibitor, wherein the production of a mRNA transcript from the DNA standard occurs in 25 the absence of the candidate inhibitor.

9. A selective classification method to identify a growth inhibitor of Candida albicans, characterized in that it comprises detecting, in the presence of a candidate inhibitor, the inhibition of the formation of a complex comprising a DNA and TFIIB pattern of the recombinant Candida albicans, in where, in the absence of the candidate inhibitor, complex formation occurs.

10. A selective classification method to identify a growth inhibitor of Candida albicans, characterized in that it comprises detecting, in the presence of a candidate inhibitor, the inhibition of the formation of a complex comprising the TFIiB of Candida albicans and the TBP of Candi da albi cans, where, in the absence of the candidate inhibitor, complex formation occurs.

11. A classification method to identify a growth inhibitor of Candida albicans, characterized in that it comprises detecting, in the presence of a candidate inhibitor, the inhibition of the formation of a a complex comprising RNA polymerase II, TBP of Ca n di da l bi cans, and PFII del Candi da a lbi can s, where, in the absence of the candidate inhibitor, complex formation occurs. 5

12. The E all of selective classification, in accordance with the claim 8, 9, 10, or 11, characterized in that the detection is carried out in the presence of a plurality of 10 candidate inhibitors in such a way that the inhibition is indicative of the inhibition caused by a candidate inhibitor of that plurality of inhibitors.

13. The method of selective classification according to claim 8, 9, 10, or 11, characterized in that they are * conduct multiple detection steps, simultaneously, using a plurality of inhibitors 20 candidates, wherein the detection of inhibition by any candidate inhibitor can be detected independently of the plurality.

14 A method for preventing the growth of Candida albicans in crops, characterized in that it comprises contacting the culture with an inhibitor that selectively inhibits the biological activity of the TFIIB of Candi da albi cans.

15. A method to prevent the growth of Candida albícans, in a mamma fero, characterized in that it comprises contacting the mammal with an inhibitor that selectively inhibits the biological activity of the TFIIB of Candi gives albícans.